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Lignocellulosic biomass is an abundant source of renewable energy, but its high recalcitrance to biodegradation needs to be overcome to allow its conversion into biofuels. Thus, pretreatment of the lignocellulosic feedstock is usually required. Fungal pretreatment using white rot fungi is an alternative process to traditional thermo-chemical pretreatments that degrades lignin and enhances the enzymatic digestibility of the lignocellulosic biomass. Fungal pretreatment can be performed in solid-state at low temperature, without added chemicals such as strong acids or bases, and no wastewater is generated. However, in comparison with traditional pretreatments, disadvantages such as long residence times, low yields, and feedstock sterilization requirements make it challenging to implement. This work investigates the fungal pretreatment of non-sterile biomass with the white rot fungus, Ceriporiopsis subvermispora, for the production of biofuels using the dedicated energy crop Miscanthus × giganteus. For this purpose, solid-state fungal pretreatment of non-sterile Miscanthus was performed in batch using Miscanthus previously colonized with the fungus as inoculum. The process enhanced the enzymatic digestibility of Miscanthus by 3- to 4-fold over that of untreated Miscanthus after 21 days of incubation time. The finished material from this non-sterile pretreatment was used as inoculum for two more generations in a sequential fungal pretreatment process. A propagation of indigenous fungi that outcolonized C. subvermispora was observed through the generations, showing that sterilization is a required step for the stability and reproducibility of fungal pretreatment. The changes in composition and structure of Miscanthus after fungal pretreatment were compared with those in corn stover, hardwood, and softwood. Fungal pretreatment increased the enzymatic digestibility of hardwood, softwood, and Miscanthus by 2 to 4.5-fold; however, it was not effective for corn stover. Also, fungal pretreatment was effective for Miscanthus harvested in winter or spring, but not for green Miscanthus harvested in fall. Therefore, fungal pretreatment with C. subvermispora showed differential effects that were feedstock-dependent.Fungal pretreated Miscanthus was used for the production of biogas via solid-state anaerobic digestion, and an increase of 25% in specific methane yield was obtained compared to the untreated Miscanthus. Fungal pretreated Miscanthus was also used for the production of fermentable sugars through enzymatic hydrolysis with commercial hydrolases for butanol production. Results indicated that fungal pretreatment with C. subvermispora does not produce significant amounts of lignocellulosic derived microbial inhibitory compounds that have been shown to inhibit butanol fermentation with Clostridium beijerinckii; thus, it does not require detoxification and/or washing after pretreatment. A techno-economic analysis of the process to produce fermentable sugars from Miscanthus using fungal pretreatment showed that the process is not feasible at full cellulosic biorefinery scale due to the high capital cost caused by the long residence time, the low bulk density of the material, and the low sugar yields.

Three studies were conducted to evaluate the fungal degradation properties of young transgenic aspen trees (Populus tremuloides). The first study focused on development of methods of decaying small diameter tree samples. The second and third studies focused on the mass loss differences and cellulose and lignin degradation between trees with different lignin types and contents. In the first study, two methods for rapid laboratory fungal decay tests of very young small diameter (5-15 mm) hybrid poplar (Populus nigra x Populus maximowiczii), yellow poplar (Liliodendron tulipifera), and willow (Salix sp.) trees were examined using the white rot fungi Trametes versicolor and Ceriporiopsis subvermispora. An agar plate method and a modified soil-agar block method were compared using non-standard size stem parts. The agar plate method did not prove to be suitable for testing materials when limited numbers of samples are available because of the extremely high variation of mass loss values. Mass loss using the modified soil-agar block technique was comparable to established methods for small and large blocks using extended colonization periods. In the second study, one-year old quaking aspen (Populus tremuloides) trees including a control wild type aspen and three lines of transgenic trees were analyzed for resistance to lignin selective white rot fungal decay. The transgenics had reduced lignin content through transfer of an antisense -4CL gene, changed syringly/guaiacyl ratio through insertion of a sense CAld5H gene, and modified lignin content and syringyl/guaiacyl through simultaneous insertion of -4CL and CAld5H genes. Mass loss was used to examine differences between genetic lines. The small diameter transgenic trees were decayed using lignin selective white rot fungus Ceriporiopsis subvermispora. A modified soil-agar block method was used with a forty day colonization time. The transgenic lines with higher S/G lignin ratio exhibited a higher mass loss percentage compared to the wild type and other transgenic lines. In the third study, the transgenic lines were analyzed for resistance to three types of fungal decay. The small diameter transgenic trees were tested using simultaneous white rot fungus Trametes versicolor, lignin selective white rot fungus Ceriporiopsis subvermispora and a brown rot fungus Poria placenta. A modified soil-agar block method was used to decay the samples. Near infrared spectroscopy and chemical analysis determined loss of cellulose and lignin variations between transgenic genotypes. Near infrared transmittance was successful in predicting the cellulose and lignin percentages of the decayed material. The reduced lignin content lines did not affect the rate of lignin decay for all fungi tested. Lignin decay rates were reduced by the increased S/G ratio lines for all fungi tested.